Human mesenchymal stem cells (hMSCs, StemCell Technologies) were cultured using Mesencult™ proliferation kit supplemented with 2mM glutamax and 1% penicillin-streptomycin (P/S). Human cardiomyocytes (CMs, PromoCell) were cultured in myocyte growth medium kit following the manufacturer’s instruction. Cells were maintained in a humidified cell culture incubator at 37°C and 5% CO2 with respective growth medium. Cell culture medium was changed every other day. All experiments were performed using hMSCs at passage 3~7 and human CMs at passages 6-9.
Mesencult proliferation kit
Manufactured by STEMCELL
Sourced in Canada, United Kingdom
The MesenCult Proliferation Kit is a cell culture media product designed to support the in vitro expansion of human mesenchymal stem/stromal cells (MSCs). The kit provides the necessary components to maintain and propagate MSCs while preserving their multi-lineage differentiation potential.
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Lab products found in correlation
Mesenpure, by STEMCELL (3 mentions)
Adipogenic differentiation kit, by STEMCELL (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Oil red o solution, by Merck Group (2 mentions)
Hmscs, by STEMCELL (1 mentions)
Egm 2 endothelial cell growth medium 2 bulletkit, by Lonza (1 mentions)
α mem medium, by Merck Group (1 mentions)
Tryple express solution, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Trypan blue solution, by Thermo Fisher Scientific (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Alcian blue 8gx solution, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Cd11b mac 1, by BD (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Mtesr1 media, by STEMCELL (1 mentions)
20 protocols using mesencult proliferation kit
1
Culturing Human Mesenchymal Stem Cells and Cardiomyocytes
2
Cell Culture Protocols for Cancer Research
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SKOV3 human ovarian cancer cells were obtained from ATCC and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin and 100 µg/mL streptomycin (P/S, Thermo Fisher, Waltham, MA, USA). Panc1 human pancreatic cancer cells and MDA-MB-231 human breast cancer cells were also obtained from ATCC and maintained in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and P/S. Primary human bone marrow stromal cells (BMSCs) derived from whole human bone marrow aspirates (Lonza, Basel, Switzerland) were cultured in the MesenCult Proliferation Kit (Stem Cell Technologies, Vancouver, Canada). Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and maintained in the endothelial cell growth medium-2 (EGM-2) BulletKit (Lonza, Basel, Switzerland). HUVECs expressing a red fluorescence protein (RFP) reporter gene were obtained from Angio-Proteomie. Cells from 75–90% confluent monolayers were passaged using 1% trypsin in phosphate-buffered saline (PBS, Thermo Fisher, Waltham, MA, USA).
3
Isolation and Characterization of Decidua Mesenchymal Stromal Cells
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DMSC were isolated from the decidua basalis that remained attached to the placenta using previously published methods18 (link)20 (link). DMSC were cultured in α-MEM medium (Sigma-Aldrich) with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-Glutamine (Sigma Aldrich).
DMSC23 cell lines were created by human telomerase reverse transcriptase (hTERT) transduction of the primary DMSC40 (link). DMSC23 cells were maintained in culture using Mesencult Proliferation Kit (Stem Cell Technologies) which consists of Mesencult basal medium, 10% Mesencult supplement, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cell cultures were maintained at 37 °C with 5% CO2 in a humidified incubator. DMSC and DMSC23 cells were characterised by flow cytometry for expression of CD90, CD146, CD166, CD44, CD73, CD105 and were negative for CD45, CD19, and HLA-DR. The cells displayed multi-lineage differentiation potential along the osteogenic, adipogenic, and chondrogenic lineages as shown previously18 (link)19 40 (link). Primary cell and cell lines were passaged after reaching 80% confluency. At each passage, cells were harvested using TrypLE Express solution (Life Technologies) and cultured on uncoated tissue culture flasks. All cultured primary cells were used for experiments up to P5 and DMSC23 cell lines up to P30.
DMSC23 cell lines were created by human telomerase reverse transcriptase (hTERT) transduction of the primary DMSC40 (link). DMSC23 cells were maintained in culture using Mesencult Proliferation Kit (Stem Cell Technologies) which consists of Mesencult basal medium, 10% Mesencult supplement, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cell cultures were maintained at 37 °C with 5% CO2 in a humidified incubator. DMSC and DMSC23 cells were characterised by flow cytometry for expression of CD90, CD146, CD166, CD44, CD73, CD105 and were negative for CD45, CD19, and HLA-DR. The cells displayed multi-lineage differentiation potential along the osteogenic, adipogenic, and chondrogenic lineages as shown previously18 (link)19 40 (link). Primary cell and cell lines were passaged after reaching 80% confluency. At each passage, cells were harvested using TrypLE Express solution (Life Technologies) and cultured on uncoated tissue culture flasks. All cultured primary cells were used for experiments up to P5 and DMSC23 cell lines up to P30.
4
Isolation and Cultivation of Bone Marrow Mononuclear Cells
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Buffy-coat units were obtained from routine processing of whole blood donated by healthy volunteers at the National Blood Transfusion Center. Isolation of circulating BMMPs was done using a gradient density media separation on Histopaque-1077 (Sigma-Aldrich, Burlington, MA, USA) by centrifugation at 500× g for 25 min at 20 °C. The isolated mononuclear cells were washed with PBS, centrifuged at 100× g for 10 min at room temperature, and resuspended in 12 mL of fresh cell culture medium (MesenCult Proliferation Kit, Stem Cell Technologies, Cambridge, UK). Antibiotics and antifungal treatment were added to the medium according to the following working solution concentrations: 50 mg/L gentamicin, 2500 μg/L amphotericin B, and 5000 units/L penicillin and streptomycin (Sigma-Aldrich, Burlington, MA, USA). BMMPs were seeded in a 75 cm2 flask and cultured at 37 °C in a humidified atmosphere and 5% CO2. Non-adherent cells were removed after 48 h. Media were replaced every 3 to 4 days until confluence was reached. BMMPs were counted and their viability was assessed with Countess TM II Automated Cell Counter using a 0.4% Trypan Blue solution (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s protocol. The assays were performed in triplicate at the first passage of the culture and before the infusion into the experimental models.
5
Multilineage Differentiation of Murine Mesenchymal Stromal Cells
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Sorted LepR-Cre/Tomato+Runx2-GFPlow stromal cells were expanded using a MesenCult proliferation Kit with MesenPure (StemCell Technologies) containing 100 U/ml and 100 μg/ml penicillin-streptomycin. Osteogenic, adipogenic and chondrogenic differentiation were induced using a Mouse Mesenchymal Stem Cell Function Identification Kit (R&D Systems). Cells were maintained with 5% CO2 in a water-jacketed incubator at 37 °C for 2–5 weeks. Mineralized osteogenic cells were identified by Alizarin Red S (Sigma-Aldrich) staining. Adipocytes were identified by characteristic production of lipid droplets and staining with an anti-FABP antibody (R&D Systems). Chondrocytic cells were identified using an Alcian Blue 8GX solution (Sigma-Aldrich).
6
Expansion and Validation of Human BMSC
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Well characterized primary human BMSC were purchased from StemCell Technologies (Cat. number: MSC-001F, Vancouver, British Columbia, Canada) and expanded using MesenCult Proliferation Kit (StemCell Technologies, Part ID 05411) following standard culture protocol. All cell culture experiments were carried out at a humidified atmosphere of 37°C and 5% CO2. For validation of the osteogenic and angiogenic gene expression changes induced by adenoviral mediated BMP2 expression in BMSC, commercially available human BMSC from two additional donors were purchased and used (henceforth referred to as donor 2 and 3 BMSC) as described in S1 Text .
7
Macrophage Response to MSC Secretome
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After confirmation of the viability of BMDMs in all wells of the 24 well plates by light microscopy, wells were washed with HBSS. Macrophage control samples (Mac ctrl) were treated with 400 µL of the DMEM based medium alone. MSC control samples (Mac MSC ctrl) contained 150 µL DMEM based medium + 250 µL plain MSC growth medium which was freshly prepared in order to rule out that any effect could come from any of the ingredients of the MSC growth medium (MesenCult proliferation kit, Stem Cell Technologies, UK). Cells in treated wells within columns 4–6 of a 24 well plate, underwent treatment with LPS for 1 h, respectively. Wells in columns 1–3 underwent change of media only. Macrophages were treated with 250 µL of the supernatants from MSCs P1-P10. Each well contained 150 µL DMEM based medium and 250 µL of MSC supernatant. Figure A3 shows a scheme of the experiment, highlighting respective treatment groups within the 24 well plates. The plates were left in an incubator at 37 °C and 5%CO2 over-night. Thereafter the macrophages were washed with HBSS and the cells from each well were lysed using 250 µL of Trizol reagent for RNA extraction.
8
Murine Mesenchymal Cell Expansion
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Mouse sorted cells were seeded at 2–3 × 103 cells per well in a 12-well adherent tissue culture plate using a MesenCult proliferation Kit with MesenPure (StemCell Technologies) containing 100 U/ml and 100 μg/ml penicillin-streptomycin. Half of the media was replaced after 7 days and at day 10, cells were stained with Giemsa staining solution (EMD Chemicals) and adherent colonies were counted.
9
Colony Quantification of Stem Cells
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Cells were seeded immediately after fluorescence assisted cell sorting at 145 cells/cm2 in a 6-well or 12-well cell culture treated dish and maintained in MesenCult Proliferation Kit (STEMCELL, Human; Catalog #05411), until colonies could be visualized under the microscope (8-12 days). Cells were washed in PBS and stained with crystal violet (3%). Colonies were identified and manually counted as clusters with greater than 30 cells forming a tight grouping.
10
Isolation of Renal CD44+ MSCs from Mice
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